New techniques have been developed as nucleic acid analysis devices for determining base sequences of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). A method utilizing electrophoresis, which is now in general use, involves preparing beforehand a cDNA (complementary DNA) fragment sample synthesized through a reverse transcription reaction of a DNA fragment or an RNA sample for sequence determination; performing a dideoxy chain termination reaction by well-known Sanger's sequencing method; thereafter performing electrophoresis for the sample; and measuring a pattern of separation and development of molecular weight to analyze the pattern.
Independently, there has been recently developed a technique of immobilizing many DNA sample fragments as samples on a support so as to determine information on sequences of the many fragments in parallel. A DNA sequencer using this technique is referred to as a massively parallel sequencer. The massively parallel sequencer performs DNA elongations on fluorescence-labeled bases as a substrate in parallel at hundreds of thousands to millions of points and detects fluorescence of reacted bases to determine a DNA nucleotide sequence. Such massively parallel sequencers are categorized as those according to a cluster-basis process and those according to a single-molecule-basis process. The respective processes will be described below.
Initially, the cluster-basis process will be described. The cluster-basis process involves analysis of clusters each of which is an amplified DNA and is in the form of a bundle of DNAs. Typically, Non-Patent Literature (NPL) 1 describes a technique including: preparing microparticles bearing DNA fragments, performing polymerase chain reactions (PCRs) on the microparticles to amplify DNA fragments into many copies, and placing the microparticles bearing PCR-amplified DNA fragments in a plate having many wells, followed by pyrosequencing-based reading. The wells each have an opening with a size equal to the size of each of the microparticles.
NPL 2 describes a technique including: preparing microparticles bearing DNA fragments, performing polymerase chain reactions on the microparticles, scattering the microparticles onto a glass support, immobilizing the microparticles thereto, performing enzymatic reactions (ligation reactions) on the glass support to allow the DNA fragments to incorporate a substrate having a fluorescent dye thereinto, detecting fluorescence emitted from the fluorescent dye, and thereby obtaining information on nucleotide sequence of each of the fragments.
Next, the single-molecule-basis process will be described. The single-molecule-basis process includes: hybridizing a labeled nucleic acid with a probe without amplification, and identifying a nucleotide sequence while elongating the nucleic acid one base by one base with nucleotides each having a fluorescent dye. This technique is reported in NPL 3. Typically, the technique described in NPL 3 is a technique of preparing a plate having many wells and arranging a nucleic acid synthetase on the plate. In this technique, fluorescence is detected while allowing nucleotides having a fluorescent dye to be incorporated into a nucleic acid to thereby elongate the nucleic acid, and thus information on nucleotide sequence of each fragment is obtained.